The structure and function of human dipeptidyl peptidase IV,possessing a unique eight-bladed beta-propeller fold

Dipeptidyl peptidase IV (DPPIV) is a serine protease, a member of the prolyl oligopeptidase (POP) family, and has been implicated in several diseases. Therefore, the development of DPPIV selective inhibitors, which are able to control the biological function of DPPIV, is important. We determined the crystal structure of human DPPIV at 2.6A resolution. The molecule consists of a unique eight-bladed beta-propeller domain in the N-terminal region and a serine protease domain in the C-terminal region. Also, the large "cave" structure, which is thought to control the access of the substrate, is found on the side of the beta-propeller fold. Comparison of the overall amino acid sequence between human DPPIV and POP shows low homology (12.9%). In this paper, we report the structure of human DPPIV, especially focusing on a unique eight-bladed beta-propeller domain. We also discuss the way for the access of the substrate to this domain.     

Elongational flow studies on conformational change in DNA induced by DNA-binding protein HU

Interaction of DNA-binding protein HU from Bacillus stearothermophilis (HUBst) with coliphage T2 DNA was investigated by observing an elongational flow-induced birefringence, Deltan, of a T2-phage DNA aqueous solution at various HU concentrations. Localized flow birefringence was observed in the pure elongational flow region, and the strain rate dependence of Deltan had a critical strain rate epsilon;(c) for the appearance of flow birefringence at all of the HU concentrations examined, indicating that a coil-stretch transition occurred at epsilon;(c) in each DNA-HU system. For strain rates larger than epsilon;(c), Deltan increased rapidly and then gradually, approaching a plateau value. The value of epsilon;(c) increased with an increase in HU concentration. Analysis based on the relationship between epsilon; (c) and the Rouse-Zimm relaxation time revealed that the increase in epsilon;(c)with increase in HU can be explained by the decrease in the size of the DNA-HU complex. The plateau birefringence value, Deltan(p), decreased at small HU concentrations but did not change at larger HU concentrations. Considering that Deltan(p) is related to the orientational order parameter of segments, it was concluded that there were at least two stages in the process of compaction of DNA induced by HU.     

Analysis of the cellular heterogeneity in the basal layer of mouse ear epidermis: an approach from partial decomposition in vitro and retroviral cell marking in vivo

In the thin epidermis, the existence of epidermal proliferation units was hypothesized. Each unit is supposed to be partitioned into each column of polygonal-shaped cornified plates, estimated to contain a central stem cell in its basal layer. We attempted to verify this hypothesis in vitro by analyzing the partially decomposed fragment of mouse ear epidermis and in vivo using retroviral cell marking. Partially decomposed fragments of the mouse ear epidermis, mostly composed of cytokeratin 14-expressing basal keratinocytes, formed multicellular colonies in vitro. They were composed of heterogeneously shaped cells, morphologically resembling the cells in each single cell-derived colony, including potential stem cells with great proliferative potency in vitro. The estimated frequency of the candidates of stem cells in the fragments was much lower than the prediction from the representative hypothesis. Retroviral cell marking with nuclear localizing LacZ protein in vivo suggested the existence of a large clonal cellular unit for epidermal renewal. From these in vitro and in vivo observations, we propose a new model for the epidermal proliferation unit.     

Cloning and characterization of a novel RING-B-box-coiled-coil protein with apoptotic function

We have identified a novel RING-B-box-coiled-coil (RBCC) protein (MAIR for macrophage-derived apoptosis-inducing RBCC protein) that consists of an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil domain, and a B30.2 domain. MAIR mRNA was expressed widely in mouse tissues and was induced by macrophage colony-stimulating factor in murine peritoneal and bone marrow macrophages. MAIR protein initially showed a granular distribution predominantly in the cytoplasm. The addition of zinc to transfectants containing MAIR cDNA as part of a heavy metal-inducible vector caused apoptosis of the cells characterized by cell fragmentation; a reduction in mitochondrial membrane potential; activation of caspase-7, -8, and -9, but not caspase-3; and DNA degradation. We also found that the RING finger and coiled-coil domains were required for MAIR activity by analysis with deletion mutants.     

Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.     

Enumeration of Legionella CFU by colony hybridization using specific DNA probes

Oligonucleotide probes and colony hybridization (CH) were applied to enumerate organisms of the genus Legionella in cooling tower water. The CH counts indicated almost the same results as CFU counts in cultivated samples derived from the water. It was concluded that it is possible to substitute the CH procedure for the conventional one.     

1H, 15N and 13C resonance assignments of yeast Saccharomyces cerevisiae calmodulin in the Ca2+-free state

Calmodulin (CaM) is a small Ca²⁺-binding protein, which has been found in all of eucaryotic cells examined. CaMs isolated from various species have highly conserved amino acid sequence (more than 90% identical), and show the same biological functions. CaM isolated from the baker's yeast (Saccharomyces cerevisiae) (yCaM), however, shares only 60% identity in the amino acid sequence with CaM from vertebrate, and shows quite distinct conformational and biochemical properties compared with those of CaM from other species. The conformational details of yCaM, however, have not been revealed yet. We achieved the chemical shift assignments of yCaM (146 amino acids) in the apo-state using uniformly ¹⁵N- and ¹³C-labeled protein. Consequently, the resonances of 95% atoms in the backbone amides were successfully assigned.     

The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.